Beta-D-Galactosidase: The highly purified Beta-D-galactosidase A will be characterized as to (a) carbohydrate composition, (b) amino acid composition, and (c) resolution of any multiple forms. In addition studies will be initiated to discern the molecular relationship between Beta-galactosidase A and B. The affinity resins (mentioned previously) will be employed in an attempt to easily obtain apparently homogeneous Beta-D-galactosidase A and B enzymes in high yields. If the affinity chromatographic procedure is successful then it will be used to purify the electrophoretic and kinetic variant of Beta- D-galactosidase from the patient with generalized gangliosidosis (Norden and O'Brien, PNAS 72:240-244, 1975). N-Acetyl-Beta-D-Glucosaminidase: The resins (a-c) will be tested in an attempt to purify both the A and B enzyme by affinity chromatography. Experiments will be carried out using H3 GM2 and H3 GA2 to characterize kinetically those cultured fibroblasts from patients known to have Tay-Sachs, juvenile GM2 and Sandhoff's diseases. Cultured fibroblasts derived from variants of the aforementioned diseases and from normal adults demonstrating an absence of synthetic hexosaminidase activity will also be examined with respect to hydrolysis of natural substrates. Alpha-L-Fucosidase: We are going to continue studies initiated this past year with alpha-L-fucosidase. 1. The relationship of the six isoelectric forms will be investigated in human liver. The forms will be separated and characterized kinetically. Interconversion of forms will be attempted by treatment with enzymes and by deamidation. The amino acid composition of the six forms will be studied. 2. The carbohydrate composition of alpha-L-fucosidase will be determined by gas liquid chromatography of trimethylsilyl derivatives of methyl glycosides. 3. The amino acid composition of purified alpha-L- fucosidase will be determined. 4. The subunit structure of alpha-L- fucosidase will be determined by sodium dodecyl sulfate gel electrophoresis. 5. Studies will be done to see when the different forms of alpha-L-fucosidase appear during development. 6. Purified alpha-L-fucosidase from liver will be used in attempts to hydrolyze the glycolipid which accumulates in fucosidotic patients. The pure enzyme will also be used to hydroly (Text Truncated - Exceeds Capacity)